Fibrotic remodeling and tissue regeneration mechanisms define the therapeutic potential of human muscular progenitors

Abstract Fibrosis is an intrinsic biological reaction toward the challenges of tissue injury that is implicated in the wound‐healing process. Although it is useful to efficiently mitigate the damage, progression of fibrosis is responsible for the morbidity and mortality occurring in a variety of diseases. Because of lacking effective treatments, there is an emerging need for exploring antifibrotic strategies. Cell therapy based on stem/progenitor cells is regarded as a promising approach for treating fibrotic diseases. Appropriate selection of cellular sources is required for beneficial results. Muscle precursor cells (MPCs) are specialized progenitors harvested from skeletal muscle for conducting muscle regeneration. Whether they are also effective in regulating fibrosis has seldom been explored and merits further investigation. MPCs were successfully harvested from all human samples regardless of demographic backgrounds. The extracellular matrices remodeling was enhanced through the paracrine effects mediated by MPCs. The suppression effects on fibrosis were confirmed in vivo when MPCs were transplanted into the diseased animals with oral submucous fibrosis. The data shown here revealed the potential of MPCs to be employed to simultaneously regulate both processes of fibrosis and tissue regeneration, supporting them as the promising cell candidates for development of the cell therapy for antifibrosis and tissue regeneration.


| INTRODUCTION
Fibrosis originates from the healing process occurring after organ injury. It is associated with excessive accumulation of extracellular matrix (ECM) that may result in remodeling of tissue parenchyma, disorganization of tissue architecture, and potential deterioration of organ function. Fibrosis can be encountered virtually in many different organs, which consequently increases the risk of mortality and morbidity in a variety of diseases. 1 Although posing major threat in diverse organ systems, fibrosis remains a major clinical challenge worldwide without effective therapies. Therefore, it is imperative to explore the potential therapeutic approaches to suppress development and accelerate regression of fibrosis.
Tissue repair and regeneration is essential for multicellular organisms because of its effect on trauma adaptation and function preservation. These biological activities occur across diverse organ systems by activation and recruitment of tissue-specific progenitor cells with potency of cell differentiation. 2 There are common sequential events between regeneration and fibrosis induced by injury. Nonetheless, the outcomes vary as a result of distinct molecular cascades and cellular interactions. 3 The predisposition is determined by the cellular ability to return to homeostatic status. Alternatively, tissue regeneration is hindered to render the progression shift to fibrosis. Once the point of the bifurcation is reached without further cellular and molecular intervention, the injured tissue may advance to steady state of fibrosis, leaving behind the likelihood of tissue generation and functional recovery.
Stem cells are competent to switch the trend toward the irreversible progression of fibrosis. 3 If the healing processes of fibrosis can be replaced by regenerative processes, a considerable impact is expected to improve health. When bone marrow derived mesenchymal stem cells (BM-MSCs) are administered, they serve as the conductor to mediate cellular responses beneficial for tissue regeneration instead of fibrosis. 4 Nonetheless, BM-MSCs also have another contradictory role in assisting generation of myofibroblasts (MFs). 5 These conflicting findings suggest that BM-MSCs may have dual roles in fibrosis. 6 On the other hand, pericytes are another cell population residing in a variety of organs with similar MSC features. They participate in injury repair with the ability of tissue regeneration, 7 but also play doubleedged function to be implicated in the development of fibrosis. 5 Although with tissue-specific properties, regulation and determination of the switch between fibrosis and regeneration in pericytes has not been completely understood. 8 The uncertainty throws a shadow when these cells are selected as the candidates of cell therapy for fibrosis resolution and tissue regeneration.
Since fibrosis is principally driven by the tissue-specific events occurring in the local microenvironment, the antifibrosis therapy is suggested to focus on tissue-specific cellular resources. 7 Muscle precursor cells (MPCs) are the tissue-specific populations of progenitors reside within muscle, an abundant tissue throughout the whole body.
After tissue injury, MPCs re-elicit cell proliferation, differentiation, migration, and tissue integration to accelerate muscle regeneration. 9 MPCs have drawn attention because of their promising therapeutic potential. They have been acknowledged for the capacity of tissue repair and regeneration. 10,11 The outcome of cell therapy confirmes their capacity of promoting functional recovery of injured tissue. 12,13 Transplantation of MPCs also successfully reconstitute the hematopoietic compartment in the BM, showing their multifunctional potential. 14 Although with confirmed results to promote tissue regeneration, the role of MPC in regulating fibrosis, which is a critical phase for tissue repair, has seldom been addressed. Regulation of fibrogenic processes is pivotal for tissue repair, and paves way for subsequent tissue regeneration. In the current study, the capacity of MPCs in regulating the tissue-specific processes of fibrosis and tissue regeneration was investigated and characterized to explore their therapeutic potential of cell therapy.

| Isolation and characterization of MPCs
Human MPCs (hMPCs) were isolated from human donors, and the freshly isolated MPCs demonstrated spindle-shaped morphology during culture and passage (Figure 1a). The MPCs were confirmed for their characteristics of expressing the markers including desmin and MyoD that were specific to the MPCs (Figure 1b). 9,12,13,15 These markers were identified in all MPCs harvested from tissue with different demographic background (Figure 1b). Formation of muscle fibers by cell fusion is an important feature of muscle differentiation and maturation. The MPCs were tested for the capacity of myofiber formation when they were induced to differentiate. All MPCs demonstrated cell alignment and fusion for myofiber generation, regardless of the tissue originating from distinct sex or gender (Figure 1c). These myofibers were positively stained for myosin heavy chain, showing muscle maturation of differentiated MPCs (Figure 1c). In addition to characterizing the cellular features of MPCs, the ability of these isolated MPCs to generate sufficient cell numbers efficiently is closely relevant to their therapeutic potential in cell therapy. To evaluate the cellular yield of MPCs, the growth curve was tested up to five passages. All MPC clones grow rapidly especially in the initial P0-P1 state with cell yields averaged to 1 Â 10 6 MPCs in both genders ( Figure 1d). Age-based comparison revealed that only P5 populations with younger-aged (less than 40-year-old) showed significantly higher cell yield compared with relative middle-aged (40-to 60-year-old) or older aged (60-80 and older than 80-year-old) counterparts ( Figure 1e). Within five passages, the cell numbers of all MPCs samples reach 1 Â 10 10 , much exceeding the numbers estimated to be required for clinical applications. 16 Next, we performed molecular profiling of MPC global gene expression to distinguish their similarity with adipose-derived stem cell (ADSC) and BM-MSC. Acquisition and analysis of gene expression profiles of other types of stem cells were accessible via Gene Expression Omnibus (GEO) series with accession codes indicated. After comparing these gene profiles, the Gene Set Enrichment Analysis (GSEA) was presented as heat maps with clustering dendrograms (Figure 1f).
The data suggested MPC retained muscle features, and expressed the molecular profiling more relevant to BM-MSC than ADSC. The consistent results were found using gene ontology (GO) term enrichment analysis in search of KEGG and gene symbol database, focusing on signaling transduction and cellular activities individually ( Figure S1).
The data indicated MPCs isolated and cultured in vitro retained original muscle characteristics while endowed with stem cell properties similar to BM-MSC.

| Regulation of fibrogenesis by MPC
To explore the effect of MPCs on fibrosis, the interaction between MPCs and fibroblasts was investigated. During fibrogenesis, fibroblasts are transformed into MFs and play major roles in the synthesis and deposition of ECM. 17 In addition to testing the fibroblasts isolated from primary culture of human tissue (hPF), the MFs derived from fibroblasts were also prepared. Identification of MFs is confirmed by the immunophenotypic criteria with the expression of α-SMA and/or vimentin without desmin. [17][18][19] They are categorized into different types such as A, V, and VA types depending on the expression of α-SMA and vimentin. 20 To avoid undesirable interference caused by cytokine supplements, the cell-density plating method was employed to prepare MFs in the current experimental approach. [21][22][23][24] During culture, α-SMA, a typical marker of MFs, progressively appeared in the cultured cells (day 6) that was not originally detected in the cultured fibroblasts (day 1) ( Figure S2a).
Quantitative analyses showed significant increases of α-SMA  of MFs, 26 which may result in undesirable biological responses and interaction during the coculture. In addition, TGF-β might induce the quiescence of myogenic stem cells 27 Accordingly, the seeding density method that had been well-recognized as the standard method to prepare MFs was employed to avoid the interference of exogenous growth factors by reagent supplement.
The coculture systems were prepared to explore the effects of MPC on hPF or MF. The trans-well experiments offering noncontacting cells that showcase only paracrine signaling was prepared.
As schematic depiction, an insert coculture system was accomplished by seeding MPCs in the lower compartments while hPF or MF were placed in the upper inserts (Figure 2a,c). To further elucidate MPC  Figure S4b). 17 Nonetheless, no expression of α-SMA was detected in MPC regardless of being cocultured with hPF or MF ( Figure S4b). In addition, expression of vimentin was originally detected in MPC. When MPC was coculture with hPF or MF, its expression decreased ( Figure S4c).
The data suggested that the original phenotypes of MPCs were not largely changed by coculture, and the possibility of the differentiation toward MF was not observed as well.
Next, we took advantage of a transcriptomic study on hPFs to investigate whether the differences of ECM remodeling were present To further explore the effects of MPCs that dictated on matrix remodeling, the expression levels of TGF-β and the tissue inhibitors of metalloproteinases (TIMPs) were investigated. Bioactive TGF-β has been confirmed to promote the pathogenic fibrotic responses and participate in creating a profibrogenic microenvironment. 34 TIMPs are natural efficient inhibitors of many MMPs to regulate proteolysis. 35 The TGFβ1, TGFβR1, TGFβR2, TIMP1, and TIMP2 gene expressions were examined through qPCR in hPF and MF. Results showed that MPC coculture generally reduced TGF-β family expression together with TIMPs levels ( Figure S5). Taken together, the data suggest that interactions of hPF or MF with MPC result in increased MMP activation and activities, and subsequently facilitating ECM remodeling.
To confirm the ability of ECM degradation directly, hPFs were cultured on the collagen-coated environment with or without MPC.
The phenotypes of hPFs were observed during coculture with MPCs using fluorescent labeling. The cocultured hPFs maintained slendershaped morphology throughout the whole culture period (Figure 6a).
It supports the observation that bidirectional communication between ECM and resident cells affects not only the surrounding matrix but also the cell phenotypes. 36 The clearance area of ECM degradation was measured in both groups. The quantitative results showed that the area of remained ECM was significantly reduced while in accordance with the elevation of ECM degradation area in the coculture

| Alteration of secretome in the coculture of fibroblasts/MFs with MPCs
The stem cells play critical roles in ECM remodeling by modulating the secretion of bioactive molecules. 38 According to the results mentioned above, we have confirmed there is a close connection between MPCs and ECM modification. However, the microenvironment of protein networks responsible for MPC effects remained elusive. To this end, the protein array encompassing essential cytokines and growth factors were analyzed in four-time repeats to compare the profiles of the conditioned media from hPF culture, with or without MPC ( Figure S7). Results of the secretory protein blots on conditioned media collected from the coculture of hPF with MPCs showed increased signals of urokinase-type plasminogen activator (uPA) and vascular endothelial growth factor (VEGF) (Figure 7a). The measurements were normalized to the profiles of hPF groups, and the foldchanges were represented as a heat map (Figure 7b, upper). For these proteins, the data from the highest to the lowest value in the distribution were further calculated for significance (Figure 7b, lower). Quantification of the two secretory proteins, uPA, and VEGF, were significantly overexpressed in the MPC cocultures compared to the hPF-alone controls (Figure 7c). To confirm the cellular origins of VEGF, real-time PCR was performed for the cultures of hPFs of MFs, with or without MPCs. The results showed that VEGF expression followed the trend demonstrated in the protein array. VEGF was found to be more highly expressed in hPF when MPCs were cocultured than in the hPF control group (Figure 7d). Intriguingly, MPC secrets more VEGF in the coculture system than culture alone (Figure 7e). In the comparison of the secretion levels of indicated cellular origins, VEGF was found to be much higher in MPCs than in hPFs (Figure 7f

| Transplantation of MPC in the animal model of oral submucous fibrosis
MPC has long been recognized for its regenerative capacity. 10,11 Transplantation of MPC into damaged sites has been documented to recover physiological function. 12

| DISCUSSION
MPCs demonstrated the capacity to simultaneously possess dual functions of facilitating fibrosis resolution and tissue regeneration, which constitute the major parts of the scenarios for tissue repair.
The analyses of molecular signature and cellular phenotypes confirmed that MPCs had the same features as satellite cells, but not for those of pericytes. 8  remodeling. VEGF directs pro-uPA activation by interacting with VEGFR2, which depends on MMP2 activation. 51,52 The activated uPA subsequently transforms the pro-enzyme plasminogen into plasmin that catalyzes the pro-MMPs into activation forms, and plasmin itself can directly target ECM for degradation. 53,54 In a positive feedback loop, uPA promotes angiogenesis by upregulating the expression of VEGFR1 and VEGFR2. 55 uPA and MMPs are implicated in ECM remodeling, 56 and these effects disappear when uPA and MMP are inhibited. 57 The mechanism of ECM remodeling of uPA relies on itself, or through generation of plasmin since activation of MMPs is required to be assisted by plasminogen. 58 MMPs enhance VEGF production, 59 and contribute to fibrosis resolution. 60 When VEGF increases in the microenvironment, it effectively stimulates the production of metalloproteinases, tissue-type and uPAs, and plasminogen activator inhibitor 1. 61 When VEGF facilitates fenestrations in endothelial cells, it increases vascular permeability, monocyte chemotaxis, and macrophages function, which are beneficial for fibrosis resolution. 60 The data presented here explain the potential mechanism accounting for the effect of reducing fibrosis by MPCs in other muscle injury models. 46 Taken together, MPCs serve as a mediator of tissue remodeling, and the effects are prominent when the fibroblasts are present.
The use of MPCs as treatment has a versatile and enormous beneficial impact on fibrosis resolution. When the therapeutic potential of VEGF, uPA and MMPs are considered, it is promising to target them in ECM remodeling to ameliorate fibrosis progression. In our secretome results, the third highest abundant soluble factor is IGFBP-3.

IGFBP-3 and VEGF are closely implicated in angiogenesis, and
IGFBP-3 is beneficial to promote VEGF secretion. 62 In addition, IGFBP-3 is demonstrated to regulate fibrosis. The reciprocal interaction between bone morphogenetic proteins and IGFBP-3 was observed in tissue remodeling by increasing MMP2. 63 A reciprocal interaction between IGFBP-3 and MMP was also found. 64,65 The role of IGFBP-3 in VEGF and MMPs regulation is important in tissue remodeling, which may be one of the mechanisms accounting for fibrosis resolution mediated by MPC.
OSF is associated with inflammation followed by subsequent fibrosis of the deep layer of the tissue under oral submucosa to cause trismus. 66 It is mediated by transformation of fibroblasts to become fibrotic-associated MFs and promote production of ECM in the oral cavity. MFs are found in the early stage of OSF, and increases correspondingly with the progression of OSF. 17 Accordingly, OSF is an appropriate model to test the capacity of MPCs by targeting MFs to ameliorate the degree of fibrosis in OSF. In OSF, the balance between MMPs and TIMPs is disturbed with increased ECM accumulation.
TIMP-1 and TIMP-2 increase in the fibroblast isolated from OSF, whereas expression of MMP attenuates. Muscle is the abundant sources with normal cells adjacent to the pathological tissue of OSF.
When MPCs were transplanted into the diseased oral submucosa, they survived in the in vivo microenvironment of OSF, differentiated toward muscle fibers, and successfully regulated MMPs for tissue remodeling and functional recovery. The data demonstrate the therapeutic potential of MPC for fibrosis. Further efforts will be required to optimize variables for clinical translation. According to our approach, it is feasible to harvest sufficient MPCs from the donor with different demographic background, which overcome the major limitation of cell availability in clinical applications. 46 Many studies have demonstrated the clinical potential of myogenic stem/progenitor cells for treating impaired muscle tissues. 67,68 Despite their therapeutic potential, the effect of tissue integration of these transplanted cells remains elusive. The studies of stem cell transplantation for the treatment of myocardial infarction has confirmed the importance of muscle integration of transplanted cells. 69,70 The heterogeneity of donor cells and inappropriate cell fusion and adhesion to local host tissue may lead to adverse tissue function. 71 Although the overall survival of transplanted cells remained low, and the integration of transplanted cells remained controversial, functional improvement was observed in many diseases after transplantation of muscular progenitor cells. 72,73 It is proposed that milieu-dependent effects may be more important than the direct differentiation and integration of the transplanted progenitor cells. 74 In the current study, the significant physiological recovery and muscle regeneration were previously published. 9,12,13,15,75 Briefly, the tissue was first minced and digested by type I collagenase 0.2% (w/v) (Sigma-Aldrich) and dispase 0.4% (w/v) (Corning) for 120 min at 37 C. After digestion, repetitive rigorous pipetting was used to release muscle fibers and fibroblasts. The tissue pellets were prepared by filtration and centrifugation, and then suspended with medium on a type I collagen coated dishes (BD Biosciences Clontech). After 24 h, the supernatant containing nonadhered MPCs was transferred to another new dish, and the original dish was used for hPFs collection. The procedures were repeated every 2 days to increase the purity of MPCs and hPFs populations. The growth medium and differentiation medium were prepared as the same as those previously described. 9,12,13,15 To prepare MFs without the interference caused by cytokine supplement in culture, the cell-density plating method was employed. 21

| Array and data analyses
The cells were harvested from the coculture and controls. Total RNA was extracted by the RNeasy Mini kit (Qiagen, Valencia, CA). The yield and quality control of mRNA samples were examined before microarray analysis. The Human Whole Genome OneArray (Phalanx Biotech, HsinChu, Taiwan) was used. Three repeats from independent specimens were enrolled. The raw data were confirmed with the Pearson correlation coefficient for technical reproducibility. Differentially expressed genes were identified when the expression was in the spot with the following criteria including a log 2 ratio ≥1 or a log 2 ratio ≤À1 and a p-value <0.05. 76 To correlate the predefined gene sets with phenotypes, data were analyzed and ranked by GSEA software from the Broad Institute with default setting. 77 The gene sets ranked by ratio were subjected to KEGG database and leading-edge analysis for uncovering genes that contribute to the enrichment scores. To validate the similarity between MPC and other mesenchymal progenitor cells, we retrieved microarray data of BM-MSC and ADSC from NCBI GEO database, and these data were further analyzed by the Multi Experiment Viewer microarray analysis software.

| Zymography
To measure the ECM proteolytic activity in the secretome harvested from the coculture of MPC and fibroblasts, zymography was applied to detect the function of MMPs. In-gel zymography was employed using the media retrieved from fibroblasts cultured with or without MPCs. The medium was centrifuged to remove cell debris, lyophilized, and resuspended in water. Equal levels of protein were determined before being loaded to each lane. MMP activity was determined by in-gel zymography using gelatin or collagen as the components to prepare the gel. After separation, gels were washed, renatured with Triton X-100, and incubated with protease inhibitors. The proteolytic bands were visualized by Coomassie brilliant blue and the densitometry was inverted for black and white to make it clear. The area digested in the gel represented the proteolytic activity of indicated MMPs with corresponding molecular weight. The regions were further quantitated by ImageJ for comparative analyses.
To directly identify the proteolytic activities of fibroblasts or MFs when culture with or without MPC, in situ zymography was applied using dye-quenched (DQ) labeled substrates (Thermo Fisher Scientific). The experiments were conducted following the protocol provided by the manufacture. The cells (1 Â 10 4 ) were seeded on the DQ-substrate (20 μg/ml) coating slides for coculture. After culture, the slides were fixed and counterstained with DAPI for the nucleus.
Fluorescence of proteolytic activity was imaged, quantified by ImageJ program, normalized and corrected by subtraction to the negative control, and quantitatively compared. 82

| Proteomic analyses
The supernatants were collected from the fibroblasts cultured with or without MPCs for 48 h, and were subjected to proteomic analyses using the Proteome Profiler Human Array (R&D, Cat. # ARY007) according to the manufacture's instruction. In brief, these samples were loaded onto the membrane spotted with the antibodies for specific markers. After overnight incubation, the results for different substrates were developed and detected. Quantification of the pixel densities was determined using AlphaEaseFC 4.0 software. Each spot was analyzed by Vision Works LS software (UVP, CA). The data were first normalized to positive control, and presented as the fold changes.

| MPCs treatment in the OSF animals
The animal model of OSF was established to evaluate the effect of MPCs in vivo. In the current model, MPCs were harvested from human samples and transplanted into the murine model as xenogeneic grafts. The chemical method reported to successfully set up OSF animal model was followed and modified using adult CB17ICR-SCID mice, with the approval of by the Animal Care and Use Committee of the institute. 39 Then, 4% phenol solution was injected into the submucosa of both sides of oral cavity by 30G-needles. One month later, MPCs were prepared (1 Â 10 7 cells/ml) and injected into the treated sites. Sham operations followed the same procedures without injection of MPCs in the OSF group. The physiological parameters of measuring the mouth opening width were recorded and compared with the control (OSF) and wide-type groups after 90 days of MPC treatment. The mouth opening was measured based on the length between the roots of the upper and lower incisors. All animals were sacrificed for tissue harvest. For histological analyses of harvested tissue, sampling of the fields for examination followed the published methodology. 83 More than 10 fields under the pathological examinations were randomly selected from each side, and at least three sections of each specimen were evaluated. Submucosal collagen accumulation was demonstrated using Masson's trichrome staining. The fibrosis scoring system that had been widely accepted for evaluating the subepithelial fibrosis was applied. 84,85 Briefly, the degree of fibrosis was scored by the histological features of collagen and associated cell density. The scores were evaluated among these groups to provide the quantitation of the fibrosis that is principally composed of collagen fibers. The existence of the muscle fiber differentiated from injected human MPCs was confirmed by RT-PCR and immunofluorescent staining using the markers derived from human origins. Expression of the MMPs in the harvested tissue was assessed by immunofluorescent staining, and the proportion of positive-stained fibroblast was quantitatively compared between the treated (MPC + OSF) and the control (OSF) groups.

| Statistics
All measurements were averaged from more than three independent experiments for indicated experimental settings. The continuous data were presented as the mean and standard deviation that were compared by t test. The categorical data were analyzed using chi-square or Fisher exact tests. In this study, the statistical analysis was calculated with GraphPad prism ver.9.3.0, and the results of charts and statistics were further confirmed by Excel and other statistic software.

| Illustration
The illustration demonstrated in the main and supplementary figures were created with Biorender.com.

| CONCLUSION
The current study confirms the novel properties of MPCs to reverse the fibrosis progression by regulating the transformation and activities of fibroblasts. In addition to being differentiated into mature muscle fibers, MPCs are competent to influence fibroblasts and remodel the fibrosis processes simultaneously after tissue injury. The data revealed the mechanism of MPC in mediating fibrosis regression through paracrine effects by regulating the activities and crosstalk of the associated molecules including MMPs, uPA, and VEGF, which promoted ECM degradation and tissue modeling. The augmented effect was further confirmed in vivo when MPCs were applied in the OSF animal models. Since MPCs could be successfully harvested from all human samples regardless of demographic background, they are demonstrated to be the promising candidates for development of the cell therapy for antifibrosis and tissue regeneration.